Sequence Elements Distal to the Ligand Binding Pocket Modulate the Efficiency of a Synthetic Riboswitch

Synthetic riboswitches can serve as sophisticated genetic control devices in synthetic biology, regulating gene expression through direct RNA–ligand interactions. We analyzed a synthetic neomycin riboswitch, which folds into a stem loop structure with an internal loop important for ligand binding and regulation. It is closed by a terminal hexaloop containing a U‐turn and a looped‐out adenine. We investigated the relationship between sequence, structure, and biological activity in the terminal loop by saturating mutagenesis, ITC, and NMR. Mutants corresponding to the canonical U‐turn fold retained biological activity. An improvement of stacking interactions in the U‐turn led to an RNA element with slightly enhanced regulatory activity. For the first position of the U‐turn motif and the looped out base, sequence–activity relationships that could not initially be explained on the basis of the structure of the aptamer–ligand complex were observed. However, NMR studies of these mutants revealed subtle relationships between structure and dynamics of the aptamer in its free or bound state and biological activity.     

Intracellular Light‐Activation of Riboswitch Activity

By combining a riboswitch with a cell‐permeable photocaged small‐molecule ligand, an optochemical gene control element was constructed that enabled spatial and temporal control of gene expression in bacterial cells. The simplicity of this strategy, coupled with the ability to create synthetic riboswitches with tailored ligand specificities and output in a variety of microorganisms, plants, and fungi might afford a general strategy to photocontrol gene expression in vivo. The ability to activate riboswitches by using light enables the interrogation and manipulation of a wide range of biological processes with high precision, and will have broad utility in the regulation of artificial genetic circuits.     

RNA-As-Graphs Motif Atlas—Dual Graph Library of RNA Modules and Viral Frameshifting-Element Applications

RNA motif classification is important for understanding structure/function connections and building phylogenetic relationships. Using our coarse-grained RNA-As-Graphs (RAG) representations, we identify recurrent dual graph motifs in experimentally solved RNA structures based on an improved search algorithm that finds and ranks independent RNA substructures. Our expanded list of 183 existing dual graph motifs reveals five common motifs found in transfer RNA, riboswitch, and ribosomal 5S RNA components. Moreover, we identify three motifs for available viral frameshifting RNA elements, suggesting a correlation between viral structural complexity and frameshifting efficiency. We further partition the RNA substructures into 1844 distinct submotifs, with pseudoknots and junctions retained intact. Common modules are internal loops and three-way junctions, and three submotifs are associated with riboswitches that bind nucleotides, ions, and signaling molecules. Together, our library of existing RNA motifs and submotifs adds to the growing universe of RNA modules, and provides a resource of structures and substructures for novel RNA design.     

Quality Control Certification of RNA Aptamer‐Based Detection

Aptamers are single‐stranded DNA or RNA molecules with a defined tertiary structure for molecular recognition. Numerous RNA aptamers with excellent binding affinity and specificity have been reported; they constitute an attractive reservoir of molecular recognition elements for biosensor development. However, RNA is relatively unstable owing to spontaneous hydrolysis and nuclease degradation. Thus, RNA aptamer‐based biosensors are prone to producing false‐positive signals. Here, we present an RNA aptamer biosensor design strategy that utilises an internal control to distinguish target binding from false‐positive signals. The sequence of a chosen RNA aptamer is expanded so that it can form three consecutive short RNA–DNA duplexes with 1) a quencher‐labelled DNA strand (Q₁DNA), 2) a dual‐fluorophore‐labelled DNA strand (F₁DNAF₂) and 3) another quencher‐labelled DNA strand (Q₂DNA). The addition of a target releases Q₂DNA from the duplex assembly, and produces the expected positive signal from F₂. However, the authenticity of target recognition is validated only if no signal is generated from F₁. We have successfully engineered two fluorescent reporters by using an RNA aptamer that binds thrombin and one that binds theophylline. Both reporters show the expected binding affinity and specificity, and are capable of reporting system malfunction when treated with nucleases and chemical denaturants. This strategy provides a simple and reliable way to ensure high‐quality detection when RNA aptamers are employed as molecular‐recognition elements.     

化工设计中常用阀门的选型方法

阀门的选型在化工管路设计中占有重要的地位,科学合理地选择阀门既能保证生产安全运行,又能降低装置的建设费用。文章介绍了闸阀、截止阀、节流阀、旋塞阀、蝶阀、球阀、止回阀、隔膜阀等常用阀门的结构及应用范围,并对化工设计中常甩阀门的选型进行了介绍与说明。     

化工设计中特殊阀门的选用说明

阀门种类繁多,结构复杂,正确选择阀门在化工管路设计中占有重要的地位。科学合理地选择阀门既能保证生产安全运行,又能降低装置的建设费用。本文介绍了疏水阀、安全阀与减压阀等特殊阀门的结构及应用范围,并对化工设计中这些特殊阀门的选型进行了介绍与说明。     

Isolation and Characterization of RNA Aptamers against a Proteasome‐Associated Deubiquitylating Enzyme UCH37

Deubiquitylating (DUB) enzymes antagonize ubiquitin‐dependent protein degradation both before and after the substrates are engaged with proteasomes. UCH37 is one of three proteasome‐associated DUB enzymes in mammals and the only protease among them from the ubiquitin carboxyl‐terminal hydrolase (UCH) family. Here, we report the identification of specific RNA aptamers for UCH37 through in vitro selection, and we describe their inhibitory effects on the DUB activity of UCH37. The RNA aptamers significantly delayed RPN13‐mediated UCH37 activation and lowered total DUB activity of proteasomes, as measured by the hydrolysis of ubiquitin‐rhodamine 110. In addition, the UCH37 aptamers efficiently facilitated the hydrolysis of peptide‐based reporter substrates of proteasomes. Thus, the UCH37 aptamers might offer a possible strategy for removing toxic cellular proteins through enhancing proteasome activity.     

双金属复合管液胀成形选材标准理论分析探讨

首先针对当前复合管液胀成形技术现状,对选材标准进行理论分析。然后通过分析衬管和基管在液胀成形复合管过程的应力应变,得到了复合管能够复合的基本要求,结合材料的力学特性,得到了衬管和基管能够实现液胀成形复合管的选材标准。最后利用有限元软件Abaqus进行模拟分析,验证了选材标准的正确性,为复合管液胀成形的初步选材提供了理论支撑,利用相关企业的实际生产,为双金属复合管液胀成形工程实际提供了重要依据。     

Endo‐β‐Glucosidase Tag Allows Dual Detection of Fusion Proteins by Fluorescent Mechanism‐Based Probes and Activity Measurement

β‐Glucoside‐configured cyclophellitols are activity‐based probes (ABPs) that allow sensitive detection of β‐glucosidases. Their applicability to detect proteins fused with β‐glucosidase was investigated in the cellular context. The tag was Rhodococcus sp. M‐777 endoglycoceramidase II (EGCaseII), based on its lack of glycans and ability to hydrolyze fluorogenic 4‐methylumbelliferyl β‐d ‐lactoside (an activity absent in mammalian cells). Specific dual detection of fusion proteins was possible in vitro and in situ by using fluorescent ABPs and a fluorogenic substrate. Pre‐blocking with conduritol β‐epoxide (a poor inhibitor of EGCaseII) eliminated ABP labeling of endogenous β‐glucosidases. ABPs equipped with biotin allowed convenient purification of the fusion proteins. Diversification of ABPs (distinct fluorophores, fluorogenic high‐resolution detection moieties) should assist further research in living cells and organisms.     

Design of a General‐Purpose European Compound Screening Library for EU‐OPENSCREEN

This work describes a collaborative effort to define and apply a protocol for the rational selection of a general‐purpose screening library, to be used by the screening platforms affiliated with the EU‐OPENSCREEN initiative. It is designed as a standard source of compounds for primary screening against novel biological targets, at the request of research partners. Given the general nature of the potential applications of this compound collection, the focus of the selection strategy lies on ensuring chemical stability, absence of reactive compounds, screening‐compliant physicochemical properties, loose compliance to drug‐likeness criteria (as drug design is a major, but not exclusive application), and maximal diversity/coverage of chemical space, aimed at providing hits for a wide spectrum of drugable targets. Finally, practical availability/cost issues cannot be avoided. The main goal of this publication is to inform potential future users of this library about its conception, sources, and characteristics. The outline of the selection procedure, notably of the filtering rules designed by a large committee of European medicinal chemists and chemoinformaticians, may be of general methodological interest for the screening/medicinal chemistry community. The selection task of 200K molecules out of a pre‐filtered set of 1.4M candidates was shared by five independent European research groups, each picking a subset of 40K compounds according to their own in‐house methodology and expertise. An in‐depth analysis of chemical space coverage of the library serves not only to characterize the collection, but also to compare the various chemoinformatics‐driven selection procedures of maximal diversity sets. Compound selections contributed by various participating groups were mapped onto general‐purpose self‐organizing maps (SOMs) built on the basis of marketed drugs and bioactive reference molecules. In this way, the occupancy of chemical space by the EU‐OPENSCREEN library could be directly compared with distributions of known bioactives of various classes. This mapping highlights the relevance of the selection and shows how the consensus reached by merging the five different 40K selections contributes to achieve this relevance. The approach also allows one to readily identify subsets of target‐ or target‐class‐oriented compounds from the EU‐OPENSCREEN library to suit the needs of the diverse range of potential users. The final EU‐OPENSCREEN library, assembled by merging five independent selections of 40K compounds from various expert groups, represents an excellent example of a Europe‐wide collaborative effort toward the common objective of building best‐in‐class European open screening platforms.     

Histone H3 N‐Terminal Peptide Binds Directly to Its Own mRNA: A Possible Mode of Feedback Inhibition to Control Translation

Give me some feedback : In vitro selection of aptamers against the H3 peptide provided specific hairpin RNAs that possess high homology with histone H3 mRNA. The identified H3 hairpin RNA binds specifically to the H3 peptide with micromolar affinity and dose‐dependently inhibits in vitro translation of the H3 protein. Consequently, the hairpin RNA and H3 peptide are one of the rare cis‐ and trans‐elements on coding regions found among housekeeping proteins in higher eukaryotes.

     

Dual pH‐ and thermal‐responsive nanocomposite hydrogels for controllable delivery of hydrophobic drug baicalein

Hydrogels have been widely used as mild biomaterials due to their bio‐affinity, high drug loading capability and controllable release profiles. However, hydrogel‐based carriers are greatly limited for the delivery of hydrophobic payloads due to the lack of hydrophobic binding sites. Herein, nano‐liposome micelles were embedded in semi‐interpenetrating poly[(N‐isopropylacrylamide)‐co‐chitosan] (PNIPAAm‐co‐CS) and poly[(N‐isopropylacrylamide)‐co‐(sodium alginate)] (PNIPAAm‐co‐SA) hydrogels which were responsive to both temperature and pH, thereby establishing tunable nanocomposite hydrogel delivery systems. Nano‐micelles formed via the self‐assembly of phospholipid could serve as the link between hydrophobic drug and hydrophilic hydrogel due to their special amphiphilic structure. The results of transmission and scanning electron microscopies and infrared spectroscopy showed that the porous hydrogels were successfully fabricated and the liposomes encapsulated with baicalein could be well contained in the network. In addition, the experimental results of response release in vitro revealed that the smart hydrogels showed different degree of sensitiveness under different pH and temperature stimuli. The results of the study demonstrate that combining PNIPAAm‐co‐SA and PNIPAAm‐co‐CS hydrogels with liposomes encapsulated with hydrophobic drugs is a feasible method for hydrophobic drug delivery and have potential application prospects in the medical field. © 2018 Society of Chemical Industry     

Polymerization Kinetics and Characterization of Dual Cured Polyurethane‐Acrylate Nanocomposites for Laminates

Summary: Four different types of montmorillonites have been dispersed by sonication at 50 °C into a propoxylated aromatic epoxy diacrylate oligomer to achieve interlayered or exfoliated nanocomposites. A thermally‐induced crosslinking reaction, forming a polyurethane network in the presence of 7 wt.‐% of a montmorillonite, has been promoted by addition of an allophanate modified polyisocyanurate based on hexamethylene diisocyanate. The kinetic behavior of the network formation has been studied at 25, 40 and 60 °C by following the disappearance of the isocyanate vibrational band found at 2 270 cm^?1. A tight crosslinked polyurethane acrylate network has been achieved by a subsequent dual UV curing promoted by a photoinitiator mixture (0.6 wt.‐%) added to the reactive mixture because of further reactions occur to the acrylate double bonds. The photopolymerization kinetic has been investigated on the different thermally treated polyurethane nanocomposite networks by Real Time FTIR spectroscopy monitoring the changes of the IR band at 810 cm^?1 assigned to the acrylate double bond vibrations. The influence of the different montmorillonite clays on the final nanocomposite morphology has been investigated by using XRD and SEM. Finally, the use of these mixtures as internal layer between two modified surface PET films has been also studied for the laminate production. The based‐PET laminate films have been characterized by determining the bending resistance and optical properties as a function of different nanofillers.

Bending resistance of the dual cured nanocomposite laminates containing 7 wt.‐% as a function of nanofiller types.     

Imaging‐Guided pHe and Glutathione Dual Responsive Polypeptide Nanogel for Smart Drug Delivery

A negatively charged polypeptide nanogel, near‐infrared (NIR) cyanine dye (Cy5.5) conjugated and 2,3‐dimethylmaleic anhydride (DMA) modified poly‐l ‐lysine‐co‐l ‐cystine (CDPLC), is synthesized and is used as an imaging‐guided sequential drug delivery system. The CDPLC nanogel can respond to two general stimulations in sequence: extracellular tumor acidic microenvironment pHe (6.8–6.5) and intracellular high concentration glutathione (GSH). Under pHe, the DMA shell of the nanogel is removed and a charge reversal takes place, resulting in positively charged nanogel which can be internalized by cancer cells easily. Once internalized into tumor cells, the increased intracellular GSH concentration further promotes DOX release from the nanogel and DOX is enriched to the nucleus. Cy5.5 is conjugated to the nanogel as an NIR fluorescent probe, making it possible for imaging‐guided drug delivery, which is confirmed by the MTT and confocal laser scanning microscopy via in vitro experiments. The as‐prepared nanogel is a potential theranostic for cancer therapy.